Obesity is associated with numerous comorbidities such cardiovascular diseases (CVD) and type 2 diabetes (T2D). The common denominator in the pathogenesis of the co-morbidities of obesity is the presence of an active, low-grade inflammatory process. In context of the recently emerging evidence on the importance of epigenetic regulation in the immune and inflammatory responses, we hypothesize that obesity induced defects in immune function is caused, at least in part, by DNA methylation changes in peripheral blood mononuclear cells in response to adipokines and cytokines secreted by adipose tissue. Based on our preliminary studies which provided strong support to this hypothesis, we aim to identify the differential methylation profiles between obese cases and lean controls and further evaluate whether these methylation changes are involved in the pathogenesis of obesity related co-morbidities. First, we will identify the CpG sites where DNA methylation differs between obese cases and lean controls employing a step-wise selection process involving 2 stages (Aim 1). After interrogation of 27,000 methylation sites in more than 14,000 genes in 100 obese cases and 100 lean controls, we will choose the most promising for validation in a subsequent stage with 300 obese cases and 300 lean controls eventually leading to the (approximately) 20 most likely/important sites. Next, we will determine the potential downstream mechanisms of DNA methylation differences on gene expression (Aim 2) by assessing whether changes in methylation of these CpG sites are associated with changes in gene expression in the cells collected from aim 1. Functional in vitro experiments will also be conducted to investigate whether demethylation of these CpG sites can activate gene expression. Furthermore, we will determine whether the effect of obesity on CVD and T2D risk factors is -at least partially- mediated by the methylation changes of these CpG sites in a total of 1600 subjects (Aim 3). Secondary specific aims will test (1) whether the above relationships are ethnicity and/or gender dependent; (2) whether 8-month aerobic exercise intervention in overweight children will change the methylation status of these differential methylation sites. Identification of methylation changes in specific genes in obesity will provide important targets for further study into the mechanisms of obesity's effect on the immune system and the potential to develop new therapies to treat multiple obesity comorbidities independent of weight loss.